The mechanisms of regulation of Acanthamoeba myosins I and II by heavy chain phosphorylation continue to be studied. The three myosin I isozymes are activated by phosphorylation of a Ser or Thr residue at a position in the actin-binding loop of the globular head which is either Glu or Asp in most other myosins. Expression of wild type and point mutants demonstrate that Glu can fully replace the phosphoSer, that the Asp mutant is 50% as active and that Ala and Asn mutants have the same low actin-activated ATPase activity as the unphosphorylated wild type. The single amino acid whose phosphorylation is essential and sufficient for full activity of the expressed catalytic domain of myosin I heavy chain kinase (MIHCK) was identified, by a novel application of mass spectrometry, as Ser627 which corresponds in position to the Ser and Thr residues whose phosphorylation is required for activity of many Ser/Thr protein kinases. S627A and S627D mutants had equally reduced activity. Interestingly, MIHCK mutants in which Thr631 (which is not phosphorylated) was replaced by Ala, Asp and Glu also had low activity while similar substitutions for Thr632 hadlittle or no effect. These data indicate that highly conserved, unphosphorylated Thr631 is essential for activity as is also Ser 627, which is phosphorylated. Previous work indicated that phosphorylation at the tip of the tail of myosin II inhibits its actin-activated ATPase activity by decreasing the flexibility of the hinge region in the otherwise coiled-coiled, alpha-helical rod. Wild type rod, a point mutant (P398A) and a deletion mutant (delta 384-408) were expressed. Electron microscopy, analytical ultracentrifugation and electric birefringence studies established that the length, shape and flexibility of rod monomers and filaments are largely dependent on the hinge region.